The Role Of Transcription Factor AflRlm1 In The Growth,Development And Pathogenicity In Aspergillus Flavus

Aspergillus flavus is a pathogenic fungi that threatens the health of animals and human beings and widely distributed in the natural environment.Aflatoxin,a secondary metabolite produced by A.flavus,is also highly pathogenic and carcinogenic.Fungal growth,development and stress response are regulated by the mitogen activated protein kinase(MAPK)signaling pathway,in which the Slt2-MPKA pathway is primarily involved in maintaining cell wall integrity.In Saccharomyces cerevisiae,the Slt2-MPKA pathway has three protein kinases consisting of Bck1,Mkk1/Mkk2 and Slt2.Rlm1 is a transcription factor downstream of Slt2-MPKA pathway and belongs to the MADS-box gene family.Protein kinase Slt2 regulates the expression of downstream genes by phosphorylating Rlm1.In this study,we identified a homologous protein of S.cerevisiae in A.flavus and named AflRlm1.Bioinformatics analysis revealed that A.flavus AflRlm1 had the highest similarity to Aspergillus oryzae Rlm1.At present,the biological function of transcription factor AflRlm1 in A.flavus has not been reported.Based on the parental strain of A.flavus CA14 PTS(Δku70,Δniad,Δpyr G),the knockout strain(ΔAflRlm1)and complementation strain(ΔAflRlm1~C)were constructed by homologous recombination.The results showed that AflRlm1 was required for vegetative growth,conidia production,sclerotium formation,aflatoxin biosynthesis and pathogenicity.The sensitivity ofΔAflRlm1 mutants to cell wall stressors(Conge Red and Calcofluor white)and cell membrane stressor(sodium dodecyl sulfate)was increased,while the ability to response to osmotic stress was decreased.Subcellular localization test revealed that the transcription factor AflRlm1 was located in the nucleus.In order to explore the regulation mechaism of transcription factor AflRlm1 by upstream protein kinase Afl Slt2,the label strains AflRlm1-HA andΔAfl Slt2-AflRlm1-HA were constructed.Western blot showed that protein kinase Afl Slt2 regulated the phosphorylation of AflRlm1.Quantitative PCR results comfirmed that the expression of AflRlm1 was regulated by upstream protein kinase Afl Slt2,while Afl Slt2 was also feedback-regulated by AflRlm1.In summary,AflRlm1 was involved in the formation of conidia and sclerotia,and the regulation of aflatoxin synthesis and pathogenicity.Meanwhile,AflRlm1 was involved in maintaining cell wall integrity and osmotic stress tolerance.The expression and phosphorylation of AflRlm1were regulated by upstream protein kinase Afl Slt2,and the expression of Afl Slt2 was also feedback-regulated by AflRlm1.This study characterized the role of transcription factor AflRlm1 in the growth,development,pathogenicity and stress response of A.flavus,which filled the gap in the research about downstream transcription factor in cell wall integrity pathway of A.flavus,and provided the possibilities for controlling aflatoxin contamination.